GTP-binding (G) proteins act as mojecular switches in signal transduction pathways that control a diversity of biological responses. Two families of G proteins have been well characterized, namely the large or heterotrimeric G proteins (subunits designated alpha,beta,gamma) and the small monomeric Ras-related G proteins. We have used the retinal G protein transducin, which is essential for vertebrate vision, as a model for large G proteins, and Cdc42, which stimulates cell-cycle progression and when hyperactive induces malignant transformation, as a model for small G proteins. In the past funding period, we used a combination of fluorescence spectroscopy and X-ray crystallography to study the molecular regulation of the GTP-binding/GTPase cycle of Cdc42, as well as how transducin and Cdc42 bind to their target/effectors. In this renewal application, we will examine two fundamentally important aspects of G protein function. The first concerns the conformational and molecular changes that the a subunit of a large G protein must undergo to exchange GTP for GDP and thus become activated for signal propagation. Using transducin as a model, we are particularly interested in determining whether the large helical domain that is adjacent to the guanine nucleotide-binding (GTPase) domain must move away from the GTPase domain in order for GTP-GDP exchange to occur and/or if other changes in the G protein a (Ga) subunit are necessary. We also want to know how the Gbetagamma subunit complex influences the activation of a Ga subunit. These studies should identify new types of activating mutations in Ga subunits that will serve as important reagents for studying other G protein signaling systems. The second major aim of the proposed studies is to understand how activated Ga subunits stimulate their target/effectors. We will determine why the Switch III domain of the a subunit of transducin (aT), which is conformationally sensitive to GTP-binding and thought to be unique to large G proteins, is essential for stimulating effector activity. We also will examine why the PDE contains two binding sites for activated aT and if there is an important regulatory interplay between these two sites. The results of these studies should raise questions regarding the necessity of a third switch domain on small G proteins like Cdc42 and the general role of target/effector dimerization in G protein signaling. We expect that the proposed work will not only be relevant to vertebrate vision but also to a variety of signaling pathways which when deregulated give rise to a number of disease states including cancer and different neurodegenerative and endocrine disorders.